Fig. 5: Linkage between the processing of premature caspase-1 and oligomeric states of extracellular ASC specks.

A Analysis of tethered caspase-1 and ASC in each fraction obtained through sucrose-gradient (5–45%) ultracentrifugation of endogenous ASC specks. Cell-free supernatants from stimulated THP-1 cells containing ASC specks were incubated with 5 μM of rB7 or rOff for 1 h, followed by sucrose-gradient ultracentrifugation (167,000×g, 10 min), and the levels of caspase-1 (Casp1) and ASC in each fraction were analyzed using western blot (top panel). Negative stain EM images of ASC specks in each fraction were obtained through sucrose-gradient ultracentrifugation (bottom panel) at the same condition as in the top panel except for the use of recombinant ASC specks. The bars indicate 100 nm. Data were representative of three independent experiments. B. Caspase-1 activation through extracellular ASC specks in the presence of varying concentrations of rB7. After various concentrations of rB7 or rOff were added to the cell media, LPS/nigericin was used to stimulate THP-1. A fluorogenic substrate (z-YVAD-AFC) was added immediately after stimulation, and caspase-1 activity was assayed by measuring the change in the fluorescence intensity. Soluble ASC in the cell-free supernatants was separated through centrifugation (18,000×g, 10 min), and the amounts of ASC were determined by band intensity on western blot. The data represent the means ± SDs from triplicate experiments. ^***p < 0.001, ^**p < 0.01, and ^*p < 0.05 compared with the control (two-tailed unpaired Student’s t-test). C Immunoblot analysis of processed caspase-1 species and mature IL-1β when cell-free supernatants from stimulated THP-1 cells were incubated with 5 μM of rB7, rOff, or DPBS (Mock) for 1 h. Data were representative of three independent experiments. D Immunoblot analysis of active caspase-1 labeled with biotin-VAD-fmk in the presence of rB7. 3 μM of rB7 or rOff were added to the extracellular space of THP-1 cells as in (B). Biotin-VAD-fmk was added 30 min before stimulation with nigericin. The labeled caspase-1 was pulled down using streptavidin-conjugated dynabead. The eluted fraction was analyzed using a western blot. “Gly” indicates the addition of glycine before stimulation to prevent pyroptotic cell lysis. Data were representative of three independent experiments. E Immunoblot of caspase-1 eluted from co-immunoprecipitation using GST-fused rB7 or rOff in stimulated cell-free supernatants. Myc-tag was fused to the C-terminus of GST-fused rB7 and rOff for detection. Data were representative of three independent experiments.